Increased intracellular concentrations of cyclic AMP (cAMP) inhibit all platelet responses including shape change, adhesion, aggregation, and secretion. The levels of cAMP are controlled at the level of receptors by stimulation or inhibition of adenylate cyclase or by its hydrolysis by cAMP phosphodiesterases (PDEs). The predominant PDE in platelets is a low Km cGMP-inhibited PDE (cGI PDE). Studies of the cGI PDE are ongoing and involve cloning and expression of the enzyme in order to study the active site region by molecular biology techniques. Previous studies from several labs indicate that this enzyme is stimulated by phosphorylation at sites distinct from the active site region. The primary objective of this proposal is to delineate the regulation of the cGI PDE in platelets and HEL cells by phosphorylation, determine the amino acid sequence of the phosphorylated site and study the structure- function relationships. The first specific aim is to compare the kinetics of the nonphosphorylated and phosphorylated forms of the enzyme both ex vivo and in vitro. The second specific aim is to determine the amino acid sequence of the phosphorylation site (s). The pattern of phosphorylation by various kinases will be compared. The final specific aim is to study the structure-function relationship of the phosphorylated site by site-directed mutagenesis. Characterization of the regulation of the cGI PDE may advance our knowledge of intracellular cell signaling mechanisms. Such insights may have direct implications in drug development in the area of selective inhibition of platelet function.